Although HIV, the causative agent of AIDS, establishes a lifelong infection that cannot be eradicated even with effective treatment, the host immune system has the ability to contain its replication for many years in which the disease remains asymptomatic. The unexpected encounter, in 1995, between the fields of HIV and chemokines has dramatically advanced our understanding of AIDS pathogenesis, opening new perspectives for the development of effective prophylactic and therapeutic measures against HIV. The first HIV-suppressive chemokines, CCL5/RANTES, CCL3/MIP-1 and CCL4/MIP-1, were identified as major components of the soluble antiviral activity produced by CD8+ T cells and were shown to act via blockade of the principal HIV coreceptor, CCR5. However, subsequent evidence has suggested the existence of other, still unrecognized, HIV-suppressive factors produced by CD8+ T cells and other cells that are activated in the course of immunologic and inflammatory responses. Owing to their inherent antiviral properties, analogues or functional mimics of antiviral chemokines are currently under development as anti-HIV therapeutics or microbicides. 1) Identification and characterization of XCL1 as a novel HIV-suppressive chemokine. Using a large cytokine array screening platform for soluble biomolecules, we identified XCL1/lymphotactin as one of the most expressed chemokines in activated CD8+ T cells. XCL1 is a member of the C (or ) chemokine family, which lack two of the four cysteines that stabilize the classic chemokine fold; hence, XCL1 is a metamorphic protein that can interconvert in solution between two alternative conformations: a classic chemokine fold, which binds and signals through the XCL1 receptor, XCR1, and an alternative fold (all-), which binds GAGs with high affinity but not XCR1. We found that XCL1 is a conformation-dependent, broad-spectrum inhibitor of HIV-1 that shows striking similarities with the biological features and antiviral mechanism of CXCL4. Analogous to CXCL4, XCL1 inhibits a broad range of HIV-1 isolates in different experimental systems, irrespective of their coreceptor-usage phenotype and genetic subtype. Furthermore, XCL1 blocks HIV-1 through a similar mechanism to that of CXCL4, mediated by direct interaction with the gp120 envelope glycoprotein. However, no specific gp120 region has hitherto been identified as critical for XCL1 binding. Of note, the antiviral activity of XCL1 is selectively associated with the alternatively-folded conformation, emphasizing again, as in the case of CXCL4, the association between HIV blockade and GAG-binding capacity. The structural and functional characterization of XCL1 and its binding site in gp120 may have relevance for the treatment and prevention of HIV-1 infection. 2) Structure-function studies on XCL1. a) To investigate the structural determinants of the HIV-inhibitory function of XCL1, we performed a detailed structure-function analysis of a stabilized all- variant, XCL1 W55D. Individual alanine substitutions of two basic residues within the 40s loop, K42 and R43, abrogated the ability of XCL1 to bind to the viral envelope and block HIV-1 infection; moreover, a loss of HIV-inhibitory function, albeit less marked, was seen upon individual mutation of three additional basic residues, R18, R35 and K46. In contrast, mutation of K42 to arginine did not cause any loss of function, suggesting that the interaction with gp120 is primarily electrostatic in nature. Strikingly, four of these five residues cluster to form a large (3502) positively-charged surface in the all- XCL1 conformation, while they are dissociated in the classic chemokine fold, which is inactive against HIV-1, providing a structural basis for the selective antiviral activity of the alternatively-folded XCL1. Furthermore, we observed that changes to the N-terminal domain, which is proximal to the cluster of putative HIV-1 gp120-interacting residues, also affect the antiviral activity of XCL1. Interestingly, the complement of residues involved in HIV-1 blockade is partially overlapping, but distinct from those involved in the GAG-binding function of XCL1. The identification of the structural determinants of antiviral activity of XCL1 may provide a basis for the design of peptide-based or small-molecule inhibitors mimicking this functional region of the chemokine. b) In collaboration with the Volkman group at the Medical College of Wisconsin, we investigated if XCL1 anti-HIV activity is dependent on access to an unfolded state. Because XCL1 unfolds and interconverts readily between its metamorphic states, stabilized variants for the monomer and dimer are needed for functional studies of each conformation. Since previous results were all obtained with the W55D variant bearing accessibility to an unfolded state, we assayed a novel XCL1 variant, CC5, that features a new disulfide bond (A36C A49C), which prevents structural interconversion by locking the chemokine into the -sandwich dimeric conformation. CC5 as well as the variants that have access to the XCL1 dimer exhibited HIV-1 inhibition, indicating that antiviral activity is associated specifically with the dimeric all- structure of XCL1 and does not require any structural interconversion or flexibility.